Leo G. Abood

Stereoselective muscarinic acetylcholine and opiate receptors in human phagocytic leukocytes

Abstract Human granulocytes and monocytes exhibited stereospecific, high-affinity binding to 3-quinuclidinyl benzilate (QNB) and dihydromorphine (DHM), specific ligands for the muscarinic cholinergic and opiate receptors respectively. The stereoselective muscarinic binding occurred in intact, viable cells as well as in cell fragments prepared by sonication. The apparent K d for the muscarinic binding in granulocytes was 16 nM, and the number of binding sites per cell was on the order of 8 × 10 4 , monocytes contained approximately 3 × 10 4 binding sites per cell with an apparent K d of 20 nM. Studies in sonicated and in non-sonicated cell preparations indicate that the affinity of the l -isomer is approximately 10 times that of the d -isomer. Both cell types also exhibited stereospecific opiate binding with an apparent K d of 10 nM for granulocytes and 8 nM for monocytes. The number of binding sites per cell was 3000 and 4000 respectively.

Phospholipid changes in synaptic membranes by lipolytic enzymes and subsequent restoration of opiate binding with phosphatidylserine

A study has been made of the role of phosphatidylserine in stereospecific opiate binding to neural membranes, utilizing specific lipolytic enzymes to attack the lipid. At very low concentrations phospholipase A2 from bee venom will preferentially hydrolyze C22:6-fatty acid; and even after a few percent of the total phosphatidylserine is hydrolyzed, opiate binding is greatly inhibited. The addition of brain phosphatidylserine will restore opiate binding; however, when the inhibition approaches 50% restoration is only partial. Exposure of membranes to phosphatidylserine decarboxylase will partially inhibit opiate binding; and the binding returns to the control level after the addition of phosphatidylserine. The partial inhibition of opiate binding by low concentrations of Triton X-100, which presumably remove lipids, can be partially reversed by phosphatidylserine. The binding of 3H-naloxone, an opiate antagonist, is similar to agonists in its behavior towards phospholipases and phosphatidylserine; however, binding of naltrexone, also an antagonist, is far less responsive. It is concluded that the phosphatidylserine associated with the opiate receptor is the C18:0, 22:6-diacyl form, which is closely associated with protein.

Stereospecific morphine adsorption to phosphatidyl serine and other membranous components of brain.

In the course of investigating various membranous components for morphine binding, it was found that the major substance responsible was phosphatidyl serine. Other acidic lipids, such as phosphatidic acid and phosphoinositides bind to a considerably lesser extent, while neutral lipids, glycolipids and other phosphatides bind slightly or not at all. Total lipid extracts from a number of regions of rat brain exhibited different degrees of binding to (-)-morphine, such regions as the cerebral cortex and thalamus being the greatest. From an examination of the pH curve for binding and the effect of ionic strength, it was concluded that the binging was largely electrostatic. The method employed was the radioactive measurement of 14C-morphine adsorption to surface films of phosphatidyl serine. When the phosphatide was dispersed in a nonionic detergent near the critical micelle concentration, adsorption was maximal, attaining a value of 1 molecule of morphine adsorbed per molecule of phosphatidyl serine in the surface micelle. The relation between binding affinity and biological potency was not consistent. Morphine adsorption occurred with films prepared from a dispersion of the phosphatide and a hydrophobic protein from synaptic membranes. With the use of levorphanol and dextrorphan it could be shown that binding of morphine was stereospecific.

Enhancement of stereospecific opiate binding to neural membranes by phosphatidyl serine.

An effort was made to elucidate the possible involvement of phosphatidyl serine in the opiate receptor by examining stereospecific 3H-dihydromorphine binding to neural membrane preparations in the presence of exogenous phosphatidyl serine. The addition of phosphatidyl serine to suspensions of either synaptic membranes or a microsomal fraction significantly enhanced both high and lower affinity binding, the Kds being 1.0 X 10(-9) and 5.7 X 10(-9) M without lipid and 5.0 X 10(-10) and 3.8 X 10(-9) M with added phosphatidyl serine. Phosphatidyl ethanolamine and lysophosphatidyl ethanolamine had an inhibitory effect on opiate binding. Sulfatides had a slight enhancing effect, while other acidic lipids and lecithin were without any effect. With the use of 3H-dansyl phosphatidyl serine it was established that sufficient exogenous lipid was associated with the membrane to account for the enhancement of opiate binding. The results were discussed from the standpoint of the modification of membrane lipids and their possible significance for the binding of opiates and other ligands. It is suggested that phosphatidyl serine may be an important component of the opiate pharmacophore.

Binding of optically pure (−)-[3H]nicotine to rat brain membranes

With the recent availability of (−)‐[3H]nicotine of high specific activity, binding studies were performed on rat brain membranes in the presence of a variety of nicotine analogues and cholinergic drugs. Both a higher affinity (K d = 2 × 10−10 M) and a lower affinity (2 × 10−9 M) site were observed; the stereoselectivity of both sites being similar. A good correlation was observed between IC 50‐values and psychotropic potency of a series of N′‐alkyl substituted nicotine analogues.

Effects of nicotinic dimethylaminoethyl esters on working memory performance of rats in the radial-arm maze

Nicotine has been found to improve memory performance in a variety of tests, including the radial-arm maze. This improvement, together with the consistent finding of a decline in cortical nicotinic receptor concentration in Alzheimers patients, has fueled the search for novel nicotinic ligands with therapeutic potential. In the current studies, a series of nicotinic compounds was tested for effects on working memory performance in the radial-arm maze. One of the three compounds tested, DMAE II (dimethylaminoethanol cyclohexyl carboxylate fumurate), produced significant improvements in working memory performance. In the first experiment, this drug produced a biphasic dose-response curve with improved performance at the 20-mg/kg dose but not at 10 or 40 mg/kg. In a second round of DMAE II administration, the same rats showed a significant improvement with the 40-mg/kg dose. In the second experiment, a new set of rats also showed a biphasic dose-response to DMAE II. The 20-mg/kg dose caused a significant improvement whereas the 40-mg/kg dose did not. Interactions of DMAE II with nicotine and mecamylamine were also studied. Nicotine (0.2 mg/kg) by itself caused a significant improvement in working memory performance. No additive effects of DMAE II with nicotine were seen. In fact, some attenuation of response was seen with the combination. Choice accuracy data for mecamylamine could not be analyzed because of excessive sedation and nonresponding. These studies show that, like nicotine, the nicotinic ligand DMAE II causes an improvement in radial-arm mace choice accuracy. The lack of additivity with nicotine may have been to the partial agonist effects of DMAE II.

Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by other unsaturated lipids

A study was undertaken on the possible involvement of phospholipids on stereospecific opiate binding to a rat brain membrane fraction comprised mainly of synaptic membranes. The addition of acidic phospholipids such as phosphatidylserine, phosphoinositides, and phosphatidic acid significantly enhanced opiate binding. With the exception of phosphatidylserine, when the acidic phospholipids contained a polyunsaturated acyl group, they were actually inhibitory, along with neutral phospholipids derived from brain. Both the C18:0, C18:1 form (derived from myelin) and the C18:0, C22:6 form of phosphatidylserine (derived from synaptic membranes) produced as much as a 45% enhancement in opiate binding. Unsaturated fatty acids were highly inhibitory, the degree of inhibition being related to the degree of unsaturation. Both phospholipase A and C were inhibitory; and the inhibitory effect of A could not be prevented by albumin or overcome with the addition of phosphatidylserine. With the use of the cross-linking agent, dinitrodifluorobenzene, it could be demonstrated that the phosphatidylserine of synaptic membranes appeared to be preferentially associated with membrane protein. The enhancement of opiate binding by phosphatidylserine diminished with increasing degree of cross-linking.

Separation of brain phosphatidylserines according to degree of unsaturation by thin-layer chromatography

Abstract Phosphatidylserines (PS) have been prepared from bovine brain using DEAE column chromatography. A method involving AgNO 3 -impragnated silica gel H thin-layer chromatography is presented for separating intact PS according to the degree of unsaturation of their fatty acids. A detailed analysis was made of the fatty acid composition of the various fractions using gas chromatography. Some data are presented on the composition of molecular species of PS in bovine brain. The two main molecular species found in cerebral cortex are tentatively assigned the structures of 1-octadecanoyl-2-docosahexaenoyl- sn -glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl- sn -glycero-3-phosphorylserine.

Barrel rotation and prostration by vasopressin and nicotine in the vestibular cerebellum

The aim of this study was to determine whether the primary sites for the action of vasopressin and nicotine in producing barrel rotation and prostration in rats were located in the modular cerebellum, i.e., lobule X. When arginine vasopressin was administered into either the fourth ventricles or directly into the nodular cerebellum via chronically implanted cannulae, the rats displayed intermittent barrel rotation and clonic convulsions. The administration of nicotine into the same areas resulted in prostration, atonia and, occasionally, clonic convulsions. A few days after the nodular cerebellum was lesioned with kainic acid, the motor disturbances resulting from either agent were virtually abolished. Histologic studies revealed that kainic acid had destroyed Purkinje and other large neurons, but had left the granular neurons relatively intact. The administration of procaine into either the fourth ventricles or nodular cerebellum blocked the behavioral responses of either vasopressin or nicotine given into the fourth ventricles. It was concluded that the nodular cerebellum is a primary site for the motor disturbances produced by vasopressin and nicotine.

Receptor binding characteristics of a 3H-labeled azetidine analogue of nicotine☆

A new radioligand, (+/-)-[3H]-1-methyl-2-(3-pyridyl)-azetidine, which is an analogue of nicotine, has been used to investigate the binding characteristics of the nicotine receptor in rat brain membranes. By Scatchard analysis, the azetidine analogue yielded a curvilinear plot with Kd values of 7 X 10(-11) and 1.7 X 10(-9) M and Bmax values of 0.3 X 10(-14) and 2.5 X 10(-14) mol/mg protein respectively. Thermodynamic analyses yielded negative free enthalpy values for both sites, a decrease in the Bmax of only the lower affinity site, and no effect on either Kd. The psychotropic potency (prostration in rats following intraventricular injection) of the azetidine analogue was about 5-fold greater than (-)-nicotine, being among the greatest of any known nicotine analogues tested to date. Since only the higher affinity Kd differed from that of (-)-nicotine, 3-fold greater, the psychotropic potency appears to be correlated with the higher affinity site. Insofar as [3H]methylcarbamylcholine, a nicotinic ligand resembling acetylcholine, exhibits a linear Scatchard with a Kd of 1 X 10(-9) M, the higher affinity site appears to be characteristic of nicotine analogues.